Agents that bind to and inhibit human cytochrome P450 2C19

ABSTRACT

The invention provides monoclonal antibodies and other binding agents to human cytochrome P450 2C19 having advantageous properties, including capacity substantially to inhibit enzyme activity of human cytochrome P450 2C19 and lack of specific binding to other human cytochrome P450s. The binding agents of the invention are useful inter alia in methods for screening drugs for metabolism by cytochrome P450 2C19, and in methods of measuring P450 2C19 levels in individuals relative to P450 2C19 levels in a control population.

BACKGROUND OF THE INVENTION

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BACKGROUND OF THE INVENTION

The human cytochrome P450s collectively metabolize a multitude of drugsand non-drug xenobiotics including toxins, mutagens and carcinogens aswell as endobiotics such as steroids, prostaglandins and fatty acids.Drug metabolism in human liver is primarily catalyzed by twelve majormicrosomal P450 enzymes having different substrate and productspecificities and are heterogeneously distributed in tissues (Ioannides,1996, CYTOCHROMES P450 METABOLIC AND TOXICOLOGICAL ASPECTS. CRC Press,New York) (Rendic and DeCarlo, 1997, Drug Metab Rev 29:413-580).P450-catalyzed metabolism of drugs and non-drug xenobiotics is a keyelement in drug disposition and may be responsible for certain adversedrug reactions, chemical toxicity and immunotoxicity (Ioannides, 1996,CYTOCHROMES P450 METABOLIC AND TOXICOLOGICAL ASPECTS. CRC Press, NewYork).

The human cytochrome P450 2C sub-family consists of four isoforms 2C8,2C9, 2C18 and 2C19. The three major alleles of P450 2C9 are the wildtype 2C9_(Arg144)(*1), 2C9_(Cys144)(*2), 2C9_(Ile→Leu359)(*3) (Haininget al. 1996, Miners and Birkett, 1998, Clin Pharmacol 45:525-538;hereinafter referred to as 2C9*1, 2C9*2 and 2C9*3 respectively). The 2Cisoforms are collectively among the most important human enzymesresponsible for the metabolism of a wide variety of drugs includingtaxol (Rahman et al., 1994, Cancer Res 54:5543-5546) phenytoin,tolbutamide, S-warfarin, losartan, S-mephenytoin and diazepam (Minersand Burkett, 1998, Clin Pharmacol 45:525-538).

Monoclonal antibodies (“MAbs”) are reagents (Yelton and Scharff, 1981,Annu Rev Biochem 50:657-680) that have proved to be of great value forthe precise identification, measurement and functional characterizationof each P450 isoform (Gelboin, 1993, Pharmacol Rev 45:413-453). The MAbsare derived from potentially immortal hybridomas and are specific andhighly inhibitory to the enzyme activity of the target P450 and thus arepowerful reagents for “reaction phenotyping” i.e., for measuring themetabolic contribution of each of the multiple P450s to a substratesmetabolism (Gelboin, 1993, Pharmacol Rev 45:413-453)

MAbs specific to seven individual major human liver (HLM) P450 isoformswere described viz., 1A1, (Fujino et al., 1982, Proc Natl Acad Sci USA79:3682-3686), 1A2 (Yang et al., 1998, Pharmacogenetics 8:375-382), 2A6(Sai et al., 1999, Pharmacogenetics 9:229-237), 2B6 (Yang et al., 1998,Biochem Pharmacol 55:1633-1640), 2D6 (Gelboin et al., 1997,Pharmacogenetics 7:469-477), 2E1 (Gelboin et al., 1996, Chem Res Toxicl9:1023-1030) and 3A4/5 (Gelboin et al., 1995, Biochem Pharmacol50:1841-1850) and to the entire 2C sub-family (Yang et al., 1998,Biochem Pharmacol 55:889-896; Park et al., 1989, Biochem Pharmacol38(18):3067-3074). The MAbs measured the contribution of each targetP450 to the metabolism of a variety of examined drugs (Yang et al.,1999, Drug Metab Dispos 27:102-109; Gelboin et al., 1999, TrendsPharmacol Sci 20(11):432-438). Many of the MAbs can also be used tomeasure the tissue P450 protein content by immunoblot analysis.

Genetic polymorphisms of cytochromes P450 result inphenotypically-distinct subpopulations that differ in their ability toperform biotransformations of particular drugs and other chemicalcompounds. These phenotypic distinctions have important implications forselection of drugs. For example, a drug that is safe when administeredto most humans may cause toxic side-effects in an individual sufferingfrom a defect in an enzyme required for detoxification of the drug.Alternatively, a drug that is effective in most humans may beineffective in a particular subpopulation because of lack of a enzymerequired for conversion of the drug to a metabolically active form.Further, individuals lacking a biotransformation enzyme are oftensusceptible to cancers from environmental chemicals due to inability todetoxify the chemicals (Eichelbaum et al., 1992, Toxicology Letters64/65, 155-122). Accordingly, it is important to identify individualswho are deficient in a particular P450 enzyme, so that drugs known orsuspected of being metabolized by the enzyme are not used, or used onlywith special precautions (e.g., reduced dosage, close monitoring) insuch individuals. Identification of such individuals may indicate thatsuch individuals be monitored for the onset of cancers.

Existing methods of identifying deficiencies in patients are notentirely satisfactory. Patient metabolic profiles are often assessedwith a bioassay after a probe drug administration. Poor metabolizers(PM) exhibit physiologic accumulation of unmodified drug and have a highmetabolic ratio of probe drug to metabolite. This bioassay has a numberof limitations: Lack of patient cooperation, adverse reactions to probedrugs, and inaccuracy due to coadministration of other pharmacologicalagents or disease effects. See, e.g., Gonzalez et al., 1994, Clin.Pharmacokin. 26, 59-70. Genetic assays by RFLP (restriction fragmentlength polymorphism), ASO PCR (allele specific oligonucleotidehybridization to PCR products or PCR using mutant/wild-type specificoligo primers), SSCP (single stranded conformation polymorphism) andTGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE(mutation detection electrophoresis) are time-consuming, technicallydemanding and limited in the number of gene mutation sites that can betested at one time.

A complication in patient drug choice is that most drugs have not beencharacterized for their metabolism by P450 2C family and othercytochromes P450. Without knowing which cytochrome(s) P450 is/areresponsible for metabolizing an individual drug, an assessment cannot bemade for the adequacy of a patient's P450 profile. For such drugs, thereis a risk of adverse effects if the drugs are administered to poormetabolizers.

Monoclonal antibodies that specifically bind to 2C family members andinhibit its activity, if available, could be used to screen drugs fortheir metabolism by 2C and/or identify 2C poor metabolizers by simplebioassays, thereby overcoming the problems in prior complicated methodsdiscussed above. However, such monoclonal antibodies represent, at best,a small subset of the total repertoire of antibodies to human cytochromeP450 2C, and have not hitherto been isolated. Although in polyclonalsera, many classes of antibody may contribute to inhibition of enzymeactivity of P450 2C family members as a result of multiple antibodies insera binding to the same molecule of enzyme, only a small percentage ofthese, if any, can inhibit as a monoclonal. A monoclonal antibody caninhibit only by binding in such a manner that it alone block orotherwise perturb the active site of an enzyme. The existence andrepresentation of monoclonal antibodies with inhibitory properties thusdepend on many unpredictable factors. Among them are the size of theactive site in an enzyme, whether the active site is immunogenic, andwhether there are any sites distal to the active site that can exertinhibition due to stearic effects of antibody binding. The only means ofobtaining antibodies with inhibitory properties is to screen largenumbers of hybridoma until one either isolates the desired antibody orabandons the task through failure.

Notwithstanding these difficulties, the present invention provides interalia monoclonal antibodies that specifically bind to human cytochromeP450 2C family members and inhibit their activity.

BRIEF SUMMARY OF THE INVENTION

The invention provides isolated binding agents that compete with amonoclonal antibody selected from the group consisting of MAb 1-7-4-8,MAb 1-5-1-3, and MAb 1-1-11-1 for specific binding to human cytochromeP450 2C19, and that specifically inhibits 2C19-catalyzed metabolism ofdiazapam and phenanthrene by at least 90%.

Preferred binding agents are monoclonal antibodies. Some binding agentslack specific binding to at least one cytochrome P450 selected from thegroup consisting of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9*1,2C9*2, 2C9*3, 2C18, 2D6, 2E1, 3A4, and 3A5. Some binding agents lackspecific binding to each of human cytochromes P450 1A1, 2A6, 2B6, 2C8,2C9*1, 2C9*2, 2C9*3, 2C18, 2D6, 2E1, 3A4, and 3A5. Preferred bindingagents are able to specifically inhibit the enzyme activity of humancytochrome P450 2C19 by at least 90%. Some binding agents are bindingfragments, such as Fab fragments.

MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1 are exemplified monoclonalantibodies. Some other monoclonal antibodies are analogs of thesemonoclonal antibodies comprising a light chain variable domain having atleast 80% sequence identity with the light chain variable domain of amonoclonal antibody selected from the group consisting of MAb 1-7-4-8,MAb 1-5-1-3, and MAb 1-1-11-1, wherein the percentage identity isdetermined by aligning amino acids in the light chain variable domainsby the Kabat numbering convention and a heavy chain variable domainhaving at least 80% sequence identity with the heavy chain variabledomain of a monoclonal antibody selected from the group, wherein thepercentage sequence identity is determined by aligning amino acids inthe heavy chain variable domains by the Kabat numbering convention.

The invention further provides cell lines producing monoclonalantibodies as described above. Cells lines can be eucaryotic orprocaryotic.

The invention further provides methods of determining whether acytochrome P450 2C family member metabolizes a compound. Such methodsentail contacting the compound with cytochrome P450 2C family member inthe presence of varying amounts of the binding agents above. Metabolismof the compound is then assayed as a function of amount of bindingagent, a decrease of metabolism with amount of binding agent indicatingthat cytochrome P450 2C family member metabolizes the compound. In somesuch methods, the compound is contacted with cytochrome P450 2C familymember in a sample containing a collection of cytochrome P450 enzymesincluding the 2C family member. A preferred P450 2C family member isP450 2C 2C19.

In some methods, the sample is a tissue sample. In some methods, thecollection of enzymes are obtained from a cell culture expressing theenzymes. In some methods, the compound is a drug, steroid or carcinogen.

The invention further provides methods of detecting cytochrome p450 2Cmembers. Such methods entail contacting a sample suspected of containingcytochrome P450 2C family member with a binding agent described above.One then determines whether the agent specifically binds to the sample,specific binding indicating the presence of the particular cytochromeP450 2C family member in the sample.

The invention further provides methods of measuring P450 2C levels in anindividual relative to P450 levels in a control population. Such methodsentail contacting a sample suspected of containing cytochrome a P450 2Cfamily member from the individual and a substrate of 2C. One thendetermines the p450 2C levels in the individual relative to P450 2Clevels in the control population.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Inhibition of diazapam and phenanthrene metabolism by monoclonalantibodies MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1.

FIG. 2. Activity of human P450 cytochromes for the metabolic conversionof S-mephenytoin to its 4, hydroxy metabolite.

FIG. 3. Monoclonal antibody combinatorial analysis of S-Mephenytoinmetabolism in human liver microsomes (HLM).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides monoclonal antibodies and other binding agents inisolated form that specifically bind to human cytochrome P450 2C19, andinhibit enzymic activity of 2C19. Preferred agents lack specific bindingto other human cytochromes P450. The invention further provides methodsof using the antibodies and other binding agents in identifyingindividuals with a deficient metabolizing 2C19 phenotype, and inscreening drugs for metabolism by cytochrome P450 2C19.

Except when noted, the terms “patient” or “subject” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals.

Specific binding between an antibody or other binding agent and anantigen means a binding affinity of at least 10⁶ M⁻¹. Preferred bindingagents bind human cytochrome P450 2C19 with affinities of at least about10⁷ M⁻¹, and preferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “antibody” includes intact antibodies, antigen-binding portionsof an intact antibody and binding fragments thereof that retain capacityto bind P450 2C19.

The terms “monoclonal antibody” (also referred to as “Mab” or “MAbs”)and “monoclonal antibody composition” refer to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodycomposition displays a single binding specificity and affinity for aparticular epitope.

The term “polyclonal antibody” refers to a preparation of more than 1(two or more) different antibodies to human cytochrome P450 2C19. Such apreparation includes antibodies binding to a range of differentepitopes.

The term “Ka”, as used herein, is intended to refer to the equilibriumassociation constant of a particular antibody-antigen interaction. Thisconstant has units of 1/M.

The term “Kd”, as used herein, is intended to refer to the equilibriumdissociation constant of a particular antibody-antigen interaction. Thisconstant has units of M.

The term “ka”, as used herein, is intended to refer to the kineticassociation constant of a particular antibody-antigen interaction. Thisconstant has units of 1/Ms

The term “kd”, as used herein, is intended to refer to the kineticdissociation constant of a particular antibody-antigen interaction. Thisconstant has units of 1/s.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a humanized immunoglobulin orthe amino acid sequence of the humanized immunoglobulin) refers to twoor more sequences or subsequences that have at least about 80%, mostpreferably 90-95% or higher nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usingthe following sequence comparison method and/or by visual inspection.Such “substantially identical” sequences are typically considered to behomologous. Preferably, the “substantial identity” exists over a regionof the sequences that is at least about 50 residues in length, morepreferably over a region of at least about 100 residues, and mostpreferably the sequences are substantially identical over at least about150 residues, or over the full length of the two sequences to becompared. As described below, any two antibody sequences can only bealigned in one way, by using the numbering scheme in Kabat. Therefore,for antibodies, percent identity has a unique and well-defined meaning.That is, percent sequence identity is the percentage of aligned aminoacids or nucleotides that are the same between two immunoglobulins ortheir coding sequences being compared.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acids according to thescheme of Kabat et al., 1987 and 1991, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.).Kabat et al. list many amino acid sequences for antibodies for eachsubclass, and list the most commonly occurring amino acid for eachresidue position in that subclass. Kabat et al. use a method forassigning a residue number to each amino acid in a listed sequence, andthis method for assigning residue numbers has become standard in thefield. Kabat et al.'s scheme is extendible to other antibodies notincluded in the compendium by aligning the antibody in question with oneof the consensus sequences in Kabat et al. The use of the Kabat et al.numbering system readily identifies amino acids at equivalent positionsin different antibodies. For example, an amino acid at the L50 positionof a human antibody occupies the equivalence position to an amino acidposition L50 of a mouse antibody.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

An isolated species means an object species (e.g., a binding polypeptideof the invention) that is the predominant species present (i. e., on amolar basis it is more abundant than any other individual species in thecomposition). Preferably, an isolated species comprises at least about50, 80 or 90 percent (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

I. Binding Agents of the Invention

A. Specificity and Functional Properties

Binding agents of the invention compete with exemplary antibodiesdesignated MAb 1-7-4-8 (ATCC PTA-3970, MAb 1-5-1-3, (ATCC PTA-3969 andMAb 1-1-11-1 (ATCC PTA-3971 for specific binding to human cytochromeP450 2C19. Production of monoclonal antibodies MAb 1-7-4-8, MAb 1-5-1-3, MAb 1-1-11-1 is described in the Examples. The data in the Examplesshow that out of the total repertoire of antibodies to human cytochromeP450 2C19, only a small proportion inhibit 2C19 enzymic activity.Binding agents that compete with MAb 1-7-4-8,MAb 1-5-1-3, and MAb 1-1-11-1 for binding to cytochrome P450 2C19 are expected to share similarinhibitory properties because inhibition by the exemplified antibodieslikely arises through binding of the exemplified antibodies to an activesite of 2C19,and competing agents bind to the same or closely proximatesite as MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1. Capacity to competewith MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1, thus defines a selectsubclass of antibodies with advantageous properties from the totalrepertoire of antibodies to human cytochrome P450 2C19.

Hybridomas producing MAb 1-7-4-8 (ATCC PTA-3970, MAb 1-5-1-3, (ATCCPTA-3969 and MAb, 1-1-11-1 (ATCC PTA-3971 have been deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209 under the Budapest Treaty and given the Accession Nos.indicated on Jan. 8, 2001. These cell lines will be maintained at anauthorized depository and replaced in the event of mutation,nonviability or destruction for a period of at least five years afterthe most recent request for release of a sample was received by thedepository, for a period of at least thirty years after the date of thedeposit, or during the enforceable life of the related patent, whicheverperiod is longest. All restrictions on the availability to the public ofthese cell lines will be irrevocably removed upon the issuance of apatent from the application.

Competition is determined by an assay in which the antibody under testinhibits specific binding of a reference antibody to an antigenicdeterminant on human cytochrome P450 2C19. Numerous types of competitivebinding assays are known for example: (See Harlow and Lane, 1988,“Antibodies, A Laboratory Manual,” Cold Spring Harbor Press). Typically,such an assay involves the use of purified human cytochrome P450 2C19,an unlabelled test antibody and a labeled reference antibody.Competitive inhibition is measured by determining the amount of labelbound to human cytochrome P450 2C19 in the presence of the testantibody. Usually the test antibody is present in excess. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as a reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to human cytochrome P450 2C19by at least 10, 25, 50, 75, 80, 90, or 95%.

Binding agents of the invention typically lack specific binding (i.e.,crossreactivity) to human cytochromes P450 other than 2C19, so that thebinding agents can be used to detect human cytochrome P450 2C19 in thepresence of other cytochromes P450. For example, binding agents of theinvention typically lack specific binding to one or more of humancytochromes P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9*1, 2C9*2, 2C9*3, 2C18, 2D6, 2E1, 3A4, and 3A5 as measured by ELISA and immunoblot. Somebinding agents of the invention, including the exemplified MAb 1-7-4-8,MAb 1-5-1-3, and MAb 1-1-11-1 lack specific binding to all of the abovehuman cytochromes P450.

As noted above, binding agents of the invention are characterized bycapacity to inhibit human cytochrome P450 2C19-catalyzed metabolism of asubstrate known to be metabolized by the enzyme. The enzyme can beassayed with any of phenacetin, 7-ethoxycoumarin, chlorzoazone orphenanthrene as the substrate (See present Examples). Assays can beperformed in either a microsome systems or a reconstituted systems ofpurified enzymes. For example, a suitable microsome system contains 1mg/mL protein of human liver microsomes or 1.6 mg protein/mL from humanlymphoblast cell lines, together with 0.2 mM substrate in a final volumeof 1.0 mL of 100 mM potassium phosphate buffer, pH 7.5, and 1 mM NADPH.An exemplary reconstituted system, in place of the microsome system,contains about 20-50 nM purified human P450 2C19, 40-100 nM cytochromeb5, 100 nM NADPH-P450 reductase, 10 μg/mL phospholipids and 0.25 mMsodium cholate. Incubations are typically carried out at 37° C. for 30min. Percentage inhibition is defined as 1-(rate of formation metabolicproduct in presence of test antibody/rate of formation of metabolicproduct in presence of control antibody), when antibody is present inexcess. (The control antibody is an antibody lacking specific binding tohuman cytochrome P450 2C19.) Some agents of the invention inhibitmetabolic capacity of isolated pure cytochrome P450 2C19 on any or allof the above substrates by at least 25%, 50%, 75%, 85%, 90% or 95% ormore.

B. Antibodies of the Invention

1. General Characteristics

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat et al., supra. An alternative structural definition has beenproposed by Chothia et al., 1987, J. Mol. Biol. 196: 901-917;, Nature,1989, 342: 878-883; and J. Mol. Biol., 1989, 186: 651-663.

2. Production

Antibodies to human cytochrome P450 2C19 can be produced by a variety ofmeans. The production of non-human monoclonal antibodies, e.g., murineor rat, can be accomplished by, for example, immunizing the animal witha preparation containing purified human cytochrome P450 or a fragmentthereof. The immunogen can be obtained from a natural source, bypeptides synthesis or preferably by recombinant expression.Antibody-producing cells obtained from the immunized animals areimmortalized and screened for the production of an antibody which bindsto human cytochrome P450 or a fragment thereof. See Harlow & Lane,Antibodies, A LABORATORY MANUAL (CSHP New York, 1988) (incorporated byreference for all purposes).

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989) and WO 90/07861 (incorporated by reference for allpurposes).

Human antibodies can be obtained using phage-display methods. See, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to humancytochrome P450 or a fragment thereof. Human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as MAb 1-7-4-8,MAb1-5-1-3, and MAb 1-1-11-1. Such antibodies are particularly likely toshare the useful functional properties of the exemplified antibodies.

3. Antibody Fragments

Antibodies of the invention include intact antibodies and fragments.Typically, these fragments compete with the intact antibody from whichthey were derived for specific binding to human cytochrome P450 2C19,and bind with an affinity of at least 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Antibody fragments include separate heavy chains,light chains Fab, Fab′ F(ab′)2, Fv, and single chain antibodiescomprises a heavy chain variable region linked to a light chain variableregion via a peptide spacer. Fragments can be produced by enzymic orchemical separation of intact immunoglobulins. For example, a F(ab′)2fragment can be obtained from an IgG molecule by proteolytic digestionwith pepsin at pH 3.0-3.5 using standard methods such as those describedin Harlow and Lane, supra. Fab fragments may be obtained from F(ab′)2fragments by limited reduction, or from whole antibody by digestion withpapain in the presence of reducing agents. (See id.) Fragments can alsobe produced by recombinant DNA techniques. Segments of nucleic acidsencoding selected fragments are produced by digestion of full-lengthcoding sequences with restriction enzymes, or by de novo synthesis.Often fragments are expressed in the form of phage-coat fusion proteins.This maimer of expression is advantageous for affinity-sharpening ofantibodies.

4. Recombinant Expression of Antibodies

Nucleic acids encoding light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding antibody chainsare operably linked to control sequences in the expression vector(s)that ensure the expression of antibody chains. Such control sequencesinclude a signal sequence, a promoter, an enhancer, and a transcriptiontermination sequence. Expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosome.

E. coli is one procaryotic host particularly for expressing antibodiesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilus, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication) and regulatory sequences such as alactose promoter system, a tryptophan (trp) promoter system, abeta-lactamase promoter system, or a promoter system from phage lambda.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

Mammalian tissue cell culture can also be used to express and producethe antibodies of the present invention (See Winnacker, 1987, FROM GENESTO CLONES (VCH Publishers, N.Y.). Eukaryotic cells are preferred,because a number of suitable host cell lines capable of secreting intactantibodies have been developed. Preferred suitable host cells forexpressing nucleic acids encoding the immunoglobulins of the inventioninclude: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293) (Graham et al., 1977, J. Gen.Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary-cells-DHFR (CHO, Urlaub and Chasin, 1980, Proc. Natl.Acad. Sci. USA 77:4216); mouse sertoli cells (TM4, Mather, 1980, Biol.Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); Africangreen monkey kidney cells (VERO-76, ATCC CRL 1587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather et al., 1982,Annals N.Y Acad. Sci. 383:44-46); baculovirus cells.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell. Calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation can be used for other cellularhosts. (See generally Sambrook et al., 1989, MOLECULAR CLONING: ALABORATORY MANUAL (Cold Spring Harbor Press, 2nd ed.) (incorporated byreference in its entirety for all purposes). When heavy and light chainsare cloned on separate expression vectors, the vectors areco-transfected to obtain expression and assembly of intactimmunoglobulins. After introduction of recombinant DNA, cell linesexpressing immunoglobulin products are cell selected. Cell lines capableof stable expression are preferred (i. e., undiminished levels ofexpression after fifty passages of the cell line).

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (See generally SCOPES, PROTEINPURIFICATION (Springer-Verlag, N.Y., 1982). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred.

5. Screening for Sequence Analogs

Many of the antibodies described above can undergo non-criticalamino-acid substitutions, additions or deletions in both the variableand constant regions without loss of binding specificity or effectorfunctions, or intolerable reduction of binding affinity (i. e., belowabout 10⁶ M⁻¹ for human cytochrome P450 2C19. Usually, the light andheavy chain variable regions of immunoglobulins incorporating suchalterations exhibit at least 80, 90 or 95% sequence identity to thecorresponding regions of a reference immunoglobulin from which they werederived, such as MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1. Preferredantibody light and heavy chain sequence variants have the samecomplementarity determining regions (CDRs) as the corresponding chainsfrom one of the above reference antibodies. Occasionally, a mutatedimmunoglobulin can be selected having the same specificity and increasedaffinity compared with a reference immunoglobulin from which it wasderived. Phage-display technology offers powerful techniques forselecting such immunoglobulins. See, e.g., Dower et al., WO 91/17271McCafferty et al., WO 92/01047; Huse, WO 92/06204.

C. Other Binding Agents of the Invention

The invention further provides nonantibody binding agents that competewith one of the exemplified antibodies for binding to human cytochromeP450 2C19. These binding agents include polypeptides, beta-turnmimetics, polysaccharides, phospholipids, hormones, prostaglandins,steroids, aromatic compounds, heterocyclic compounds, benzodiazepines,oligomeric N-substituted glycines and oligocarbamates. Largecombinatorial libraries of the compounds can be constructed by theencoded synthetic libraries (ESL) method described in Affymax, WO95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051,Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which isincorporated by reference for all purposes). Peptide libraries can alsobe generated by phage display methods. See, e.g., Devlin, WO 91/18980.The libraries of compounds are screened for binding to human cytochromeP450 in competition with one of the reference antibodies MAb 1-7-4-8,MAb 1-5-1-3, and MAb 1-1-11-1.

II. Human Cytochrome P450 2C19

The cDNA for human cytochrome P450 2C 19 has been cloned, sequenced, andexpressed (see e.g., Goldstein et al., 1991, Biochemistry 30:3247-55).Sources of other cytochromes P450 (e.g., for use in testing for lack ofcrossreactivity) are described by Nebert, 1991, DNA & Cell Biol. 10,1-14; Nelson et al., 1996, Pharmacogenetics 6, 1-42). Insect cells(e.g., SF9) with appropriate vectors, usually derived from baculovirus,are also suitable for expressing 1A2 and other cytochromes P450. SeeLuckow et al., 1988, Bio/Technology 6:47-55; Gonzalez et al., 1991,Meth. Enzymol., 206, 93-99 (incorporated by reference for all purposes).Other expression systems include yeast (Ellis et al., supra), E. coli(Gillam et al., 1995, Archives Biochem. Biophys. 319, 540-550; vacciniavirus (Gonzalez, 1989, Pharmacol. Res. 40, 243, and human AHH-1lymphoblastoid cells (Crespi et al., 1989, Carcinogenesis 10, 295-301).

Humans shows a wide range of 2C19 activities. Variations of P450activity in a tissue can result from a variety of factors includingnutritional factors, chemical inducers in the environment, age, sex andgeneral physiological or disease condition of the subject individual.MAbs to 2C19 can define the P450 variations related to genetic, age,sex, nutritional and environmental influences on P450 metabolism. Therole in the metabolism of a drug by a single or multiple P450s can beimportant in drug discovery for understanding P450 dependent partnerdrug relationships that can be toxic. The P450s can be major controlelements of the metabolic rates of drug metabolism as well as theirpharmacologic character. The MAbs can also identify drugs toxic due tothe absence of a polymorphic P450. This information can lead to betterunderstanding of P450 activity in drug choice, dosage and efficacy.

Several therapeutically important compounds are metabolized by 2C19. Thelist includes S-mephenytoin, diazepam, diclofenac, mephenytoin,tolbutabmide, bufuralol, imipramine, omaprazole, hexobarbital, andproguanil.

III. Methods of Use

A. Identifying Compounds Metabolized By P450 2C19

Binding agents of the invention that inhibit enzymic activity of humancytochrome P450 2C19 can be used to assay whether compounds aremetabolized by 2C19. Compounds include xenobiotics, such as a currentlyused and new drugs, carcinogens, pesticides or other industrial orenvironmental chemicals, or any endobiotic, such as a steroid E hormone.The assay can indicate not only that a compound is metabolized by 2C19but also the contribution of 2C 19 to metabolizing the compound relativeto other cytochromes P450 present in microsomes or cell homogenates.

Assays are performed by contacting a compound under test with humancytochrome P450 2C19 in reaction mixtures containing varying amounts ofa binding agent of the invention. For example, two separate reactionsmay be set up, one in which the binding agent of the invention ispresent, and the other, a control in which the binding agent is absent.The human cytochrome P450 is often present as a microsomal extract fromhuman or animal cells or cell lines or an extract from cell culturesexpressing a collection of recombinant P450s including 2C19. The assayis performed under conditions in which 2C19 is known to be active onknown substrates, such as phenanthrene (See Examples). Metabolism of thecompound under test is then followed from the disappearance of thecompound or appearance of a metabolic product of the compound as afunction of time (e.g., nmol product/sec). See, e.g., Buters et al.,1994, Drug Metab. Dispos. 22: 688. The metabolism of the compound isanalyzed as a function of the amount of binding agent present. If themetabolism quantitatively decreases with amount of binding agent, it canbe concluded that 2C 19 metabolizes the compound.

The percentage inhibition of 2C19 metabolism of a test compound mayreflect both the inherent efficiency of a binding agent in blocking 2C19activity and the contribution of cytochromes P450 other than 2C19 inmetabolizing the compound. The inherent blocking efficiency of a bindingagent can be determined by measuring inhibition of metabolism in areaction mixture in which only 2C19 is present, or alternatively, in areaction mixture in which a collection of cytochromes P450 are presentbut the substrate is known to be metabolized only by 2C19. Comparison ofthe percentage inhibition determined in these circumstances with thepercentage inhibition of metabolism of a test substrate when a mixtureof cytochromes P450 are present indicates the relative contributions of2C19 and other enzymes in the mixture to metabolism of the testsubstrate. For example, if metabolism of a control substrate by pure2C19 is inhibited by a binding agent by 90% and metabolism of a testsubstrate by a mixture of cytochromes P450 including 2C19 is inhibited45%, it can be concluded that in the mixture, 2C19 contributes about45/90=50% of metabolizing activity on the test substrate. Binding agentshaving a high degree of inhibition (e.g., at least about 90%) of a knownsubstrate are particularly effective for quantitative analysis asdescribed above.

The anti 2C19 Mab can also be used to identify substrates metabolized byCYP2C19 in human liver tissues. Recognition of the nature andcontribution of CYP2C19 in individuals can permit studies of drug-druginteractions based on competitive metabolism. MAb 1-7-4-8, MAb 1-5-1-3,and MAb 1-1-11-1 can also be used as reagents for CYP2C19 basedmetabolism studies of procarcinogens and promutagens. These MAbs canalso be used in drug metabolism studies for understanding drugdisposition, activation and therapeutic applications.

Information made available by the above methods can be exploited in anumber of applications. Drugs determined to be processed by 2C19 shouldin general not be prescribed to patients deficient in 2C19 metabolism,or should be prescribed in reduced amounts or with close monitoring.Particular caution is needed in combination therapies involving twodrugs metabolized by the 2C19 pathways. The information can also bevaluable in drug design and screening. That is drugs can be designed orscreened such that they are metabolized to a significant extent byseveral P450 enzymes, and are not therefore likely to cause side effectsin those deficient in any single enzymes. Recognition that a carcinogenor other environmental toxin is deactivated by 2C19 signals thatdeficient metabolizers are at particular risk from the carcinogen orcompound. Conversely, recognition that a carcinogen or otherenvironmental toxin is activated to harmful form by 2C19 indicates thatdeficient metabolizers are less prone to harm from exposure to such acompound relative to extensive metabolizers.

B. Use of Agents for Diagnosing P450 Metabolic Variations

The binding agents are useful diagnostics to determine a patient'smetabolic profile prior to treatment with a drug known or suspected tobe metabolized by 2C19. Patients identified as defective in 2C19metabolism can be given alternative therapy, a lower dosage oradditional monitoring to avoid damaging side effects from their DMphenotype. Diagnosis can be performed as described below.

1. Binding Assay

Binding agents of the invention are useful for the quantitativemeasurement of the amount of individual P450 proteins in a sample, whichmay contain multiple forms of other P450 proteins. Binding betweenbinding agent and cytochrome P450 2C19 in the sample can be detected byradioimmunoassay, ELISA or immunoblotting (see Harlow and Lane, supra).The type of immunoassay can be tailored to the particular application.In radioimmunoassay, the binding agent of the invention is typicallylabeled. In ELISA, the binding agent is typically unlabelled anddetected using a secondary labeled reagent with affinity for the bindingagent (e.g., anti-IgG ³⁵S-or ³H-labeled MAb). Immuno blots areparticularly useful for screening a sample with a panel of antibodies todifferent cytochromes P450.

These assays can be tailored to measure P450 2C19 levels in anindividual relative to P450 2C19 levels in a control population. Themethod entails contacting a sample suspected of containing cytochromeP450 2C19 from the individual with a 2C19 substrate. One then determinesthe P450 2C19 levels in the individual relative to the 2C19 levels in acontrol population.

Other Uses

The binding agents of the invention can also be used for affinitypurification of cytochrome P450 2C19. The basic procedure for affinitypurification requires only one or two steps and can yield highlypurified milligram quantities of cytochrome P450 2C19. For example, thebinding agent can be covalently bound to Sepharose™, which is made intothe form of either column or a slurry for batch purification. A samplecontaining cytochrome P450 2C19 is them passed through the column orslurry and binds to the binding agent-linked Sepharose™. The nonboundmaterial containing unrelated proteins and cytochromes P450 other than2C19 are thoroughly eluted leaving the cytochrome P450 2C19, which canthen be eluted and used for a variety of chemical and physical studies.See, e.g., Cheng et al., 1948, J. Biol. Chem. 259, 12279-12284.

Monoclonal antibody based immunohistochemical methods can be applied tolocalize and examine the distribution cytochrome P450 2C19 afterdifferent inducer administration, during various physiological statesrelated to nutrition, age, and sex, and in different species andtissues. Furthermore, the intracellular distribution of the cytochromeP450 2C19 can be determined in a way not possible by standardbiochemical methods which generally cannot identify the presence ofspecific forms of cytochrome P450 proteins in isolated tissues andorganelles. See, e.g., Gelboin, 1993, Pharmacol. Rev. 45, 413-453.

Variations of P450 activity in a tissue can result from a variety offactors including nutritional factors, chemical inducers in theenvironment, age, sex and general physiological or disease condition ofthe subject individual. MAbs to 2C19 can define the P450 variationsrelated to genetic, age, sex, nutritional and environmental influenceson P450 metabolism. The role in the metabolism of a drug by a single ormultiple P450s can be important in drug discovery for understanding P450dependent partner drug relationships that can be toxic. The P450s can bemajor control elements of the metabolic rates of drug metabolism as wellas their pharmacologic character. The MAbs can also identify drugs toxicdue to the absence of a polymorphic P450. This information can lead tobetter understanding of P450 activity in drug choice, dosage andefficacy.

EXAMPLES Materials and Methods

Chemicals

Tolbutamide, diclofenac, diazepam, phenanthrene and NADPH were obtainedfrom Sigma (St. Louis, Mo.). Taxol (paclitacel), 6α-hydroxytaxol,S-mephenytoin, 4′-hydroxymephenytoin, bufuralol, 1′-hydroxybufuralol,hydroxytolbutamide and 4′-hydroxydiclofenac were obtained from GentestCorporation (Woburn, Mass.). Imipramine and desipramine were obtainedfrom ICN Biomedicals Inc. (Aurora, Ohio). All reagents were ofanalytical grade. Nordiazepam was a gift from Dr. Shen Yang.2-hydroxyimipramine was a gift from Novartis Pharmaceuticals.

P450 Expression and Hybridoma Production

P450 2C9 recombinant baculovirus was previously constructed (Grogan etal., 1995) with cDNA P450 2C19 (a kind gift from Dr. Joyce Goldstein,NIEHS, NIH, Research Triangle Park, N.C.) and cDNA P450 2C8 (Kimura etal., 1987) and were inserted into baculovirus transfection vectors(MaxBac 2.0 transfer vectors and Bac-N-Blue transfection kit,Invitrogen, Inc., Carlsbad, Calif. ). The resulting recombinantbaculovirus was used to express the P450s in Sf9 (Spondopterafrugipedra) cells. Cells (1.0×10⁶/mL) were infected with the recombinantvirus (MOI=1-5) and supplemented with 1 μg/mL heme-albumin complex(Grogan et al., 1995). Three days post infection, cells were havested,the P450 difference spectrum measured, and the cells were stored at −80°C. The P450 was extracted from the cells as previously described(Gelboin et al., 1998). Yielding 268.0 nanomole P450 2C8, 22.5 nanomoleP450 2C9, and 60.5 nanomole P450 2C19 each from one liter of individualcell culture preparations.

The extracted expressed P450 was used as the immunogen. Balb/c mice wereimmunized with 30-50 μg of extracted P450 2C8, 2C9 or 2C19 emulsified in0.2 mL adjuvant once a week for three weeks, sacrificed and the spleencells isolated. The complete immunization technique and hybridization ofspleen cells with myeloma cells (NS-1) has been detailed (Gelboin etal., 1998). Three mice were used for each immunization. The resultinghybridomas were screened by ELISA using the same extracted materialdescribed above. Extracted material from SF9 cells infected with wildtype baculovirus was used to test for specific binding of the antibodiesto the immunogen. Hybridomas producing antibodies that bound to wildtype material were discarded.

The cDNA expressed human P450 enzymes used for immuno-assays andmetabolic inhibition assays were obtained from vaccinia infected HepG2cells (Battula et al., 1987) (Gonzalez and Korzekwa, 1995), Supersomes(Gentest, Woburn, Mass.), and/or Baculosomes (Panvera Corporation,Madison, Wis.). Human liver samples were provided by the CooperativeHuman Tissue Network (CHTN) funded by the National Cancer Institute.Microsomes from human liver were prepared as described (Alvares et al.,1970), and total P450 content measured by difference spectra (Omura andSato, 1964). Protein content was measured with BCA Protein Assay Kit(Pierce, Rockford, Ill.) using BSA as a standard.

Immunoassays

ELISA was performed by a generally used method (Harlow and Lane, 1988).96-well plates were coated with 1.5 picomole of P450 enzyme. Culturefluid and/or diluted ascites was used as the primary MAb, with analkaline phosphatase conjugated anti-mouse IgG (Fc specific) (JacksonImmuno-Research, West Grove, Pa.) used as a secondary antibody.Colorimetric assays were measured at 405 nm wavelength as detailed(Krausz et al., 2000). Wells with an optical density (O.D.) reading ≧0.6with specific immunogen and an O.D. ≦0.1 with wild type antigen wereinvestigated further. All resulting specific hybridomas were cloned atleast three times. SDS-PAGE gels and immunoblots (IB) were performed aspreviously described (Gelboin et al., 1996). MAb isotyping was performedusing radial immunodiffusion kits purchased from “The Binding Site” (SanDiego, Calif.). All MAbs described are mouse IgG₁, with the exception ofMAb 1-68-11, which is a mouse IgM.

Metabolic Incubations

Metabolism and immunoinhibition studies were performed using cDNAexpressed P450 enzymes and human liver microsomes (HLM). Metabolicincubations were generally conducted as follows: 0.057 to 30 μl (1.2 to1000 μg) of ascites protein containing MAb was pre-incubated with 30-50pmole of expressed P450 enzyme or 150-300 pmol P450 in HLM in 0.5 mL ofbuffer (100 mM Tris pH 7.5) at 37° C. for 5 minutes. Substrate (in 10 μlmethanol), 1 mM NADPH, and additional buffer were added to a finalvolume of 1.0 mL. The reactions were allowed to proceed for 60 minuteswith the tolbutamide, and S-mephenytoin; 15 minutes with diclofenac and30 minutes with phenanthrene, diazepam, imipramine, and bufuralol andstopped with 1 mL acetone. The metabolites were extracted with 8 mLdichloromethane and subjected to HPLC separation conditions fordiclofenac (50 μM) to 4′-hydroxydiclofenac (Crespi and Penman, 1997),tolbutamide (200 μM) to hydroxytolbutamide (Relling et al., 1990),S-mephenytoin (150 μM) to 4′-hydroxymephenytoin (Heyn et al., 1996),diazepam (150 μM) to nordiazepam (Yang et al., 1998c), imipramine (200μM) to desipramine and 2-hydroxyimipramine (Yang et al., 1999), taxol(150 μM) to 6α-hydroxytaxol (Rahman et al., 1994), phenanthrene (200 μM)to 9,10-diol and 9-phenol (Yang et al., 1999), and bufuralol (50 μM ) to1′-hydroxybufuralol (Gelboin et al., 1997) are previously described.HPLC was performed using a Hewlett Packard Model 1050 Series systemequipped with an autosampler, a quaternary solvent delivery system, anda dioarray detector controlled by the Hewlett Packard Chemstationssoftware. Metabolite retention times were compared with authenticstandards (when available) and metabolite peaks were quantitated basedon ratios with internal standards. Control incubations contained an MAbagainst hen egg white lysozyme (MAb HyHel-9) to assess for nonspecificreactions. Percent of inhibition was calculated based on activity in thepresence of the specific MAb relative to the activity with the controlMAb. Percent inhibition equals 100 minus % of control. Data is the meanof duplicate incubations using expressed enzymes, and triplicateincubations using HLM.

Inhibition of phenanthrene metabolism to 9,10-diol was used to test forinhibitory cross reactivity of the MAbs with expressed P450 1A1, 1A2,2B6, 2C8, 2C9, 2C18, 2C19, 2E1, 3A4, and 3A5; inhibition of phenanthrenemetabolism to the 9-phenol was assayed for cross-reactivity with P4502A6; and inhibition of bufuralol metabolism to 1′-hydroxybufuralol wasassayed for cross-reactivity with P450 2D6. Inhibition of diazepamconversion to nordiazepam (NDZ) was also used to test the inhibitorycross-reactivity of the MAbs among the P450 2C isoforms.

Isolation and Screening of Hybridomas and Monoclonal Antibodies

MAbs to P450 2C8

MAbs from 830 hybridoma clones produced 16 MAbs that bound to 2C8. Threeof the 16 hybridoma clones produced MAbs that bound specifically andstrongly to only 2C8. The three MAbs were 281-1-1, 13-1-3 and 5-1-5which were both specific and strong inhibitors (90%) of expressed 2C8enzyme activity. The three MAbs also immunoblot 2C8.

MAbs specific to individual P450 2C9, 2C19 and to isoform sets of the 2Csub-family

Screening for MAbs to 2C9: MAbs produced from 1088 hybridomas werescreened and 68 clones were identified that bound to 2C9. Six of the 68bound strongly and inhibited 2C9 enzyme activity. Further screeningresulted in the isolation of MAb 763-15-5, specific and highlyinhibitory to 2C9 at low levels of MAb. MAbs from other clones wereisolated and characterized and found to bind and inhibit unique sets of2C sub-family isoforms as detailed in the Results below. The binding ofa MAb to more than one isoform detects an epitope common to the varioustarget isoforms. (See Results, Tables 1-7).

Another MAb resulting from immunization with 2C9*2 was MAb 292-2-3. Thedetails of its isolation have been previously described (Krausz et al.,2000) and its further characterization are described in the Results.Previously Mab 1-68-11 was made with purified rat liver 2c/RLM5 (Park etal., 1989) which was subsequently found to inhibit all four of the human2C isoforms, 2C8, 2C9, 2C18 and 2C19 (Gelboin et al., 1997)

Screening for MAbs to 2C19: The screening for binding of MAbs formedfrom 763 hybridoma clones detected the binding to 2C19 MAbs from 15 toclones. Three of the latter MAbs strongly and specifically bound andinhibited 2C19 metabolic activity. These are MAb 1-7-4-8, MAb 1-5-1-3and MAb 1-1-11-1 and are further discussed below.

Results

The specificity of MAb 1-7-4-8, MAb 1-5-1-3, and MAb 1-1-11-1 wasdetermined with expressed P450 2C19 as measured by ELISA binding (Table1). The three MAbs bind specifically and strongly to P450 2C19 asmeasured by ELISA and do not bind to the twelve other drug metabolizinghuman P450 members (P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9*1, 2C9*2,2C9*3, 2C18, 2D6, 2E1, 3A4, and 3A5) (see Table 1 below).

TABLE 1 Specificity of Monoclonal Antibody Binding to Expressed P4502C19 as Measured by ELISA Mab/ 1A1, 1A2, 2A6, 2B6, P450 2C8 2C9*1 2C9*22C9*3 2C18 2C19 2D6, 2E1, 3A4, 3A5 MAb 1-7-4-8 0.0⁺ 0.14 0.0 0.15 0.01.20 0.0 MAb 1-1-11-1 0.0 0.20 0.14 0.12 0.0 1.41 0.0 MAb 1-5-1-3 0.00.19 0.28 0.11 0.0 1.16 0.0 ⁺Values equal absorbance at 405 nm.

Mab 1-7-4-8, Mab 1-5-1-3, and Mab 1-1-11-1 do not immunoblot (IB) theirtarget P450 2C19. The mmetabolism of two substrates of the 2C familymembers, phenanthrene (Shou et al., 1994; see Table 2A below) ordiazepam (see Table 2B below) were used to measure the inhibitoryactivity of Mab 1-7-4-8, Mab 1-5-1-3, and Mab 1-1-11-1

Table 2A and B Specificity of Inhibitory Activity of MAbs Anti-P450 2C19for Phenanthrene (A) and Diazepam (B) Metabolism

TABLE 2A % INHIBITION OF PHENANTHRENE{circumflex over ( )} METABOLISM2C9*1 2C9*2 P-450 1A1 1A2 1B1 2A6 2B6 2C8 Arg₁₄₄ Cys₁₄₄ 2C18 2C19 2D62E1 3A4 3A5 MAbs 1-7-4-8 —** — — — — — —^(#) —^(#) — 97.6 — — — — Anti-1-1-11-1 —  — 7.8 — — — —^(#) —^(#) — 97.5 — — — — 2C19 1-5-1-3 —  — — —— — —^(#) —^(#) — 97.5 — — — — {circumflex over ( )}formation of 9-10diol was measured except for 2A6, where 9-phenol was measured, and 2D6,where bufurolol was used as the substrate and 1-OH bufurolol wasmeasured. **(−) indicates <1.5% inhibition. ^(#)the MAbs specificallyand strongly inhibit (>95%) P450 2C19 at optimal MAb concentration. Athigher levels of MAb (greater than 4-fold) there is a slightcross-reaction with P450 2C9. (See FIG. 1)

TABLE 2B % INHIBITION OF DIAZEPAM⁺ METABOLISM P-450 1A1 1A2 1B1 2A6 2C19MAbs 1-7-4-8  —** —^(#) —^(#) — 97.6 Anti-2C19 1-1-11-1 — —^(#) —^(#) —97.5 1-5-1-3 — —^(#) —^(#) — 97.5 ⁺formation of diazepam N-demethylation(NDZ) was measured. **(−) indicates <1-10% inhibition. ^(#)the MAbsspecifically and strongly inhibit (>95%) P45 2C19 at optimalconcentration. At higher levels of MAb (greater than 4-fold) there is aslight cross-reaction with P450 2C9. See FIG. 1

The data in Table 2A and B show the inhibitory activity of the MAbstoward the 2C family-catalyzed metabolism. The inhibition ofphenanthrene metabolism for 2C19 was greater than 97%. The inhibition ofdiazepam metabolism was also greater than 97%. The three MAbs are thussensitive and precise probes for measuring 2C19-catalyzed metabolism inliver and other tissues.

MAb 1-7-4-8 is an extremely strong inhibitor of 2C19 enzyme activitywhen added even at a low level of 0.5 μl of ascites fluid (>95%) (seeFIG. 1). MAb 1-7-4-8 shows slight inhibitory cross reactivity to 2C9,less than 20% when added at a very high level of 6.0 μl of ascites.(FIG. 1, Tables 1 and 2). Similar cross-reaction was observed with MAb1-1-11-1 and MAb 1-5-1-3.

Thus, at very high concentrations, there is a slight (up to 30%)inhibition of P450 2C9. However, this cross-reactivity is eliminated and95% inhibition of P450 2C19 is maintained when optimal concentrations ofthe MAbs are used.

The amino acid sequence identity among the P450 2C family (2C8, 2C9,2C18 and 2C19) is greater than 77%, with 2C9 and 2C19 homology at 91%.Thus the subject MAbs detect and target P450 2C19, an isoform closelyrelated structurally to the other 2C isoforms. The extraordinaryspecificity of the MAbs is unique and not exhibited by any otherantibody or chemical probe.

The single and the combinatorial use of the MAbs can “reactionphenotype”, i.e., determine the metabolic contribution andinterindividual variation of a P450 isoform for the metabolism of a drugor non-drug xenobiotic in human liver microsomes. The utility of the MAbbased analytic system was examined with the model substrates: taxol,diazepam, tolbutamide, diclofenac, mephenytoin and imipramine. The MAbsystem can identify drugs metabolized by a common P450 or several P450sand polymorphic P450s. The MAb system identifies drugs or drug metabolicpathways that are catalyzed by a single P450 and thus can be used for invivo phenotyping. The MAb system can identify whether a particular drugis metabolized by a single P450 that may exhibit polymorphic expressionin humans. The MAb system offers large potential for studies ofcytochrome P450 function useful in Drug Discovery and reduce thepossibility of adverse drug reactions due to polymorphisms and druginteractions.

FIG. 2 shows the activity of fourteen expressed human P450s for themetabolic conversion of S-mephenytoin to its 4-hydroxy metabolite.S-mephenytoin has been used as a model substrate for the in vivophenotyping of 2C 19 (Streetman et al., 2000). S-mephenytoin (100 μM)was incubated with 30-50 pmol of c-DNA expressed P450 in 1 mL ofphosphate buffer containing 1 mM NADPH. The reaction was incubated for 1hr. at 37° C. 4′-Hydroxymephenytoin was assay by HPLC as described inMaterials and Methods. Data are the mean of duplicate incubations.

FIG. 3 shows MAb combinatorial analysis of S-mephenytoin metabolism inhuman liver microsomes. MAb 1-7-4-8 was an exemplary MAb utilized inthis study. FIG. 3 shows also that 2C19 is the dominant enzymemetabolizing mephenytoin in HLM contributing about 80% of itsmetabolism. MAbs were added as indicated to 300 pmol of P450 in humanliver microsomes (HLM) and pre-incubated for 5 min. at 37° C. Thereaction was initiated by addition of S-mephenytoin (100 μM) and NADPH(1 mM) and allowed to proceed for 60 min. The metabolite was assayed byHPLC as described in material and methods. Hy-Hel, a MAb to lysozyme,was used as a control. Each bar represents the inhibition observed bythe addition of the MAb(s) indicated and is relative to the barpreceding it. The 2C19 was the predominant expressed isoform exhibitinghydroxylase activity which was fifteen fold greater than the activity of2C9* 1. The combinatorial addition of anti-2C9 and anti 2C19 showed, incontrast to the relative activities of the expressed enzymes, that 2C9had a significant role in mephenytoin metabolism, contributing about10-20% of activity in the eight HLM. The indication that 2C9 exhibits arole for mephenytoin metabolism suggests that mephenytoin metabolism isnot an absolute measure for in vivo 2C19 phenotyping.

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Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1. An isolated antibody or fragment thereof that competes with amonoclonal antibody selected from the group consisting of MAb 1-7-4-8(ATCC PTA-3970), MAb 1-5-1-3 (ATCC PTA-3969), and MAb 1-1-11-1 (ATCCPTA-3971) for specific binding to human cytochrome P450 2C19,and thatspecifically inhibits 2C19-catalyzed metabolism of diazapam, mephenytoinand phenanthrene by at least 85%.
 2. The isolated antibody or fragmentthereof of claim 1 that is a monoclonal antibody.
 3. The isolatedantibody or fragment thereof of claim 1 that lacks specific binding toat least one cytochrome P450 selected from the group consisting of humancytochromes P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9*1, 2C9*2, 2C9*3,2C18, 2D6, 2E1, 3A4, and 3A5.
 4. The isolated antibody or fragmentthereof of claim 1 that lacks specific binding to each of humancytochromes P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9*1, 2C9*2, 2C9*3,2C18, 2D6, 2E1, 3A4,and 3A5.
 5. The isolated antibody or fragmentthereof of claim 1 that specifically inhibits the enzyme activity ofhuman cytochrome P450 2C19 by at least 90%.
 6. An isolated antibody orfragment thereof of claim 1 that is MAb 1-7-4-8 (ATCC PTA-3970) or abinding fragment thereof.
 7. An isolated antibody or fragment thereof ofclaim 1 that is MAb 1-5-1-3 (ATCC PTA-3969) or a binding fragmentthereof.
 8. An isolated antibody or fragment thereof of claim 1 that isMAb 1-1-11-1 (ATCC PTA-3971) or a binding fragment thereof.
 9. Themonoclonal antibody of claim 2 that is a Fab fragment.
 10. Themonoclonal antibody of claim 2 that is a mouse antibody.
 11. A cell linethat produces the monoclonal antibody of claim
 2. 12. The cell line ofclaim 11 that is an eucaryotic cell line.
 13. The cell line of claim 11that is a procaryotic cell line.
 14. The monoclonal antibody of claim 2comprising a light chain variable domain, wherein the light chainvariable domain comprises three CDR regions from the light chain of amonoclonal antibody selected from the group, and the heavy chainvariable domain comprises three CDR regions from the heavy chain of amonoclonal antibody selected from the group.